Imidazole, iminodiacetic acid sepharose 6B, lipoamide, electrophoresis reagents, and NAD
were obtained from Sigma-Aldrich (St. Louis, USA). Isopropyl-β-D-thiogalactopyranoside (IPTG) was purchased from Promega (Madison, USA). Dihydrolipoamide was synthesized by the reduction of lipoamide using sodium borohydride. The primers and dNTP were purchased from Bioneer (Daejeon, Korea). The Muta-Direct
Site-Directed Mutagenesis Kit was supplied by iNtRON Biotechnology (Seongnam, Korea). Ni-NTA His-Bind Resin was acquired from QIAGEN (Hilden, Germany). The
XL1-Blue containing a human E3 expression vector, pPROEX-1:E3, was a generous gift from Dr. Mulchand S. Patel of the University at Buffalo, the State University of New York.
- Site-Directed Mutagenesis
Site-directed mutagenesis was performed using a mutagenesis kit (iNtRON Biotechnology, Sungnam, ROK) with two mutagenic primer pairs, as listed in
. The mutations were confirmed by DNA sequencing.
Primers for the site-directed mutagenesis. The mismatched bases are underlined
Primers for the site-directed mutagenesis. The mismatched bases are underlined
- Expression and Purification of the Human E3 Mutant
Three mL of an overnight culture of
XL1-Blue containing the human E3 mutant expression vector was used to inoculate 1 L of LB medium containing ampicillin (100 μg/mL). The cells were grown at 37 ℃ to an absorbance of 0.7 at 595 nm, and IPTG was then added to a final concentration of 1 mM. The growth temperature was shifted to 30 ℃ and the cells were allowed to grow overnight. The overnight culture was harvested by centrifugation at 4000×g for 5 min. The cell pellets were then washed with a 50 mM potassium phosphate buffer (pH 8.0) containing 100 mM NaCl and 20 mM imidazole (Binding buffer), and then recollected by centrifugation at 4000×g for 5 min. The pellets were resuspended in 10 ml of Binding buffer. The cells were lysed by sonication and centrifuged at 10,000×g for 20 min.
The supernatant was loaded onto a Ni-NTA His-Bind Resin column that had been washed with 2 column volumes of distilled water and then equilibrated with 5 column volumes of Binding buffer. After loading the supernatant, the column was washed with 10 column volumes of Binding buffer and then with the same volume of Binding buffer containing 50 mM imidazole. The E3 mutant was eluted with Binding buffer containing 250 mM imidazole. The E3 mutant was then eluted with Binding buffer containing 250 mM imidazole and then dialyzed three times against 50 mM potassium phosphate buffer (pH 8.0) containing 0.25 mM EDTA to remove the imidazole.
- SDS-Polyacrylamide Electrophoresis
SDS-PAGE analysis of the proteins was performed in 12% SDS-PAGE gel. The gel was stained with Coomassie blue after electrophoresis.
- E3 Assay
The kinetic parameters were determined by an E3 assay performed at 37 ℃ in a 50 mM potassium phosphate buffer (pH 8.0) containing 1.5 mM EDTA with various concentrations of the substrates, dihydrolipoamide and NAD
. The activity was recorded spectrophotometrically by observing the reduction of NAD
at 340 nm using a SPECORD200 spectrophotometer (Analytik Jena AG, Jena, USA). One unit of activity is defined as 1 μmol of NAD
reduced per min. The data was analyzed using the SigmaPlot Enzyme Kinetics Module (Systat Software Inc., San Jose, USA).
- Spectroscopic Study
The UV-visible spectra were recorded using a SPE-CORD200 spectrophotometer, and the data from 325 nm to 550 nm was transferred to an ASCII file. The spectra were then drawn using the MicroCal Origin program (Photon Technology International, South Brunswick, USA). The fluorescence spectra were recorded using a FP-6300 spectrofluorometer (Jasco Inc., Easton, USA). The samples were excited at 296 nm and the emissions were recorded from 305 nm to 580 nm. The data was transferred to an ASCII file and the spectra were drawn using the MicroCal Origin program.
RESULTS AND DISCUSSION
Site-directed mutagenesis has been used for structure-function studies of human E3 and other proteins.
The site-directed mutagenesis with the mutagenic primers listed in
resulted in the construction of the mutant E3 expression vectors. The entire DNA sequence of the human E3 coding region was sequenced to confirm the integrity of the DNA sequences. No other mutations were found except for the site-directed mutations at residue-50. The mutants were expressed in
and purified on a Ni-NTA His-Bind Resin column. The purification steps were followed by SDS-PAGE (
). The gel (12%) confirmed the purity of the mutants. An E3 assay was performed, as described in the
, and the data was analyzed using the SigmaPlot Enzyme Kinetics Module (Systat Software Inc., San Jose, USA).
lists the kinetic parameters and the free energy difference (D
) for the mutant and wild-type human E3s. The
values of both mutants were reduced by approximately 2000-fold, suggesting that Cys-50 is very important to the efficient catalytic step of the conversion of the substrates to products. The
values toward the dihydrolipoamide of the mutants decreased by 3- to 4-fold, indicating that the mutations improve the efficiency of enzyme binding to dihydrolipoamide. Cys-50 is located the end of the dihydrolipoamide binding channel, as shown in
. The location of Cys-50 may be responsible for these changes in the
values toward the dihydrolipoamide of the mutants. The
value toward NAD
of the C50A mutant increased 2.5-fold, indicating that the mutation makes enzyme binding to NAD
less efficient, whereas that of the C50T mutant was similar, suggesting that the mutation does not have a significant effect on enzyme binding to NAD
. The catalytic efficiency (
) of the C50A mutant toward NAD
decreased 5317-fold, indicating that the mutation makes the enzyme substantially inefficient to NAD
. This corresponds to the destabilization of the transition state of human E3 catalysis by approximately 5.3 kcal mol
. The catalytic efficiency of C50T mutant toward NAD
was reduced 2057-fold, indicating that the mutant became substantially inefficient toward NAD
. This corresponds to destabilization of the transition state of human E3 catalysis by approximately 4.7 kcal mol
. The results suggest that the conservation of Cys-50 in human E3 is very important to the efficient catalytic process of the enzyme.
SDS-PAGE gel (12%) for the purification of C50T mutant E3. Lane 1, molecular weight markers (from top to bottom, β-galactosidase 116.3 kDa, bovine serum albumin 66.2 kDa, ovalalbumin 45.0 kDa, lactate dehydrogenase 35.0 kDa, REase Bsp981 25 kDa, β-lactoglobulin 18.4 kDa, lysozyme 14.4 kDa); lane 2, supernatant; lane 3, flow-through; lane 4, Binding buffer containing 50 mM imidazole; lane 5, Binding buffer containing 250 mM imidazole; lane 6, previously purified recombinant human E3 as a control.
Steady state kinetic parameters and the free energy difference (ΔG) for mutant and wild-type human E3s. The E3 assay was performed at 37 ℃ in a 50 mM potassium phosphate buffer (pH 8.0) containing 1.5 mM EDTA. Values are mean of three independent determinations
aDihydrolipoamide.bCalculated from the equation: ΔG = −RT ln((kcat/Km)mutant/((kcat/Km)wild-type using kcat/Km values for NAD+ as described in references 10 and 11. cNot applicable.
Homodimeric structure of human E3. Two subunits are shown as cartoons, representing secondary structures in a single color (red and green, respectively). FAD (yellow), NAD+ (blue) and Cys-50 (cyan) are shown as sticks and spheres, respectively. The arrows indicate the dihydrolipoamide binding channels. The structure was drawn using the PyMOL program (DeLano Scientific LLC). The PDB ID for the human E3 structure is 1ZMC.
The changes in the spectroscopic properties of the mutants were examined by UV-visible absorption and fluorescence spectroscopy. Human E3 contains a single FAD as a prosthetic group in each subunit. Therefore, it shows the characteristic UV-visible absorption spectrum for flavoproteins, as shown in
(dotted line). The overall shapes of the C50A mutant (dashed line) and C50T mutant (solid line) spectra were similar to that of the wild-type human E3 spectrum (dotted line) but with two noticeable differences. First, the ratios (2.6 for C50A mutant and 2.1 for C50T mutant) between the absorbance of the valley (near 390 nm) and peak (near 450 nm) of the mutants were significantly smaller than those of the wild-type enzyme (3.4). Second, the spectra of the mutants were blue-shifted by 4 nm for the C50A mutant and 10 nm for the C50T mutant, indicating that the environment of FAD becomes more hydrophilic due to these mutations. This agrees well with the nature of these mutations, which are expected to provide a more hydrophilic environment near FAD of human E3.
Spectroscopic properties of human E3s. (a) UV-visible spectra of the C50A mutant (dashed line), the C50T mutant (solid line) and wild-type (dotted line) human E3s. The spectra were recorded using a SPECORD200 spectrophotometer, and the data from 325 nm to 550 nm was transferred to an ASCII file. The spectra were then drawn using the MicroCal Origin program. (b) Fluorescence spectra of the C50A mutant (dashed line), the C50T mutant (solid line) and wild-type (dotted line) human E3s. The enzymes were excited at 296 nm and the emissions were observed from 305 nm to 575 nm. The data was transferred to an ASCII file and the spectra were then drawn using the MicroCal Origin program.
Two fluorescence emissions were observed when human E3 was excited at 296 nm, as shown in
. The first emission from 305 nm to 400 nm was mainly Trp fluorescence. The second emission from 480 nm to ＞ 550 nm was assigned to FAD fluorescence. The Trp fluorescence was quenched due to fluorescence resonance energy transfer (FRET) from Trp to FAD. A comparison of the fluorescence spectra of E3s revealed a significant difference in the ratio between the relative intensities of the first and second fluorescence emissions. The ratios (1.8 for C50A mutant and 0.7 for C50T mutant) between the relative intensities of the first and second fluorescence emissions of the mutants were much smaller than those of the wild-type enzyme (4.9). This suggests that FRET from Trp to FAD was disturbed significantly in the mutants, indicating that the mutations affected the environment of FAD in human E3 so energy transfer from Trp residues to FAD was severely affected. Cys-50 is located very close to FAD within the van der Waals distance, as shown in
. The Cys-50 to Ala or Thr mutations will result in a more hydrophilic environment near FAD. The mutations also destroy the active disulfide center between Cys-45 and Cys-50, which restricts the freedom of Cys-50. This destruction will impart freedom to the substituted amino acids at residue-50, which can also be responsible for the altered spectroscopic properties.
In this study, the effects of Cys-50 to Ala and Thr mutations in human E3 on the catalysis and structure of the enzyme were examined by site-directed mutagenesis, E3 activity measurements and spectroscopic methods. The Cys-50 to Ala and Thr mutations in human E3 result in substantially reduced
values, indicating that the conservation of Cys-50 is very important for the efficient catalytic function of the enzyme. The mutations make enzyme binding to dihydrolipoamide more efficient. This suggests that Cys-50 is involved in enzyme binding to dihydrolipoamide. This is consistent with the location of Cys-50, which is located at the end of the dihydrolipoamide binding channel. The catalytic efficiencies toward NAD
of the mutants were also deteriorated severely, indicating that the mutations significantly destabilize the transition state of human E3 catalysis by 5.3 kcal mol
(C50A mutant) and 4.7 kcal mol
(C50T mutant). The spectroscopic studies of the mutants suggest that the environment near FAD becomes more hydrophilic as a result of these mutations so the UV-visible spectra of the mutants are blue-shifted and the efficient FRETs between Trp and FAD are deteriorated.
The author thanks Dr. Mulchand S. Patel (University at Buffalo, the State University of New York) for a generous gift of anE. coliXL1-Blue containing a human E3 expression vector. The author is grateful to Dr. Tai Jong Kang (Daegu University) for providing a fluorometer. This research was supported in part by the Daegu University Research Grant.
van den Heuvel L.
Goebel H. H.
Eur J. Pediatr.
DOI : 10.1007/s00431-003-1282-z
Williams C. H.